In developmental biology it is understood that three-dimensional forces are critically important in tissue differentiation. In vitro investigations now suggest that micro-mechanical forces acting upon individual cells are equally as important for their phenotypic differentiation. Since endothelial cells in vivo are supported by the nanometer-micron structure of the subendothelial extracellular matrix (ECM), the micro- mechanical forces which act upon these cells must also be exerted at this nanoscale. Investigating the role of these micro-mechanical forces is difficult since ECMs are not only structurally complex but also contain multiple proteins and other bioactive constituents. The central aim of this proposal is to examine the effect of ECM topography on cell behavior using 3-D synthetic polymeric replicas of this highly complex surface. Replicas are produced using a lost-wax-like casting technique providing macromolecular accuracy. Since they may be entirely synthetic, this permits experimental separation of the structural-mechanical from the biochemical influences acting upon cells. It hypothesized that 3-D synthetic vascular prosthetics which replicate the ECM nano- scale will be useful in improving cellular differentiation and hence the antithrombotic properties of endothelialized prosthetics. The first aim is to systematically investigate ECM anatomical variations in vessels which vary greatly in the mechanical stresses placed upon endothelial cells, including arteries and veins of differing diameters, flow conditions (shear, pulsatility, turbulence) and compliance. This will provide insight into the in vivo role of matrix morphology and can serve to guide choices for the appropriate matrix morphology for different vascular biomaterial applications. Anatomical studies will use ECM replicas since these permit macromolecular level analysis of fine structure while also preserving the orientation of fine structure with gross vascular anatomy, unlike conventional microscopic methods. This will also permit study of ECM morphology in, for example, regions of stasis and turbulence at bifurcations. The second aspect of these studies will examine endothelial cells adherent to matrix replicas of differing morphology, under both static and shear conditions. Evaluations will examine mechanical aspects of cell adhesion (cytoskeletal structure, integrin receptor expression and location), and markers of phenotypic differentiation, gene expression, and antithrombotic properties (cFOS, various mRNAs, prostacyclin production, platelet interaction) to elucidate the relative role of matrix morphology and shear stresses on endothelial cell behavior.